Controlling Peptide Structure and Function

Abstract

The function of a protein or peptide is governed by its unique secondary structure and intrinsic dynamic properties. Hence, an ability to control secondary structure and an understanding of its dynamic behavior would allow for the regulation of protein function. However, proteins exhibit conformational complexity, which impedes a thorough investigation of the relationship between structure and function. Thus, model peptides present as ideal candidates for this purpose. Research undertaken in this thesis aims to control the secondary structure and hence, peptide activity, in addition to probing the intrinsic dynamic properties corresponding to specific structural changes within peptides. This research deepens our understanding of the these fundamental behaviors potentially transcending to proteins, which may provide important insights into their functions.Chapter one provides an overall introduction to the importance of secondary structure in proteins/peptides, and the structural dynamic behavior that governs protein function. Various strategies are introduced to control the secondary structure and antibacterial activity of peptides, along with the single-molecule junction technique used to probe the structural dynamic properties of peptides. Chapter two presents the photopharmacological approach to regulate secondary structure, and hence, antibacterial activity of a series of gramicidin S peptide mimetics through the photochemical control of a component azobenzene photoswitch. Detailed 1H NMR spectroscopy and density functional theory (DFT) calculations were used to define the secondary structure in the cis and trans configurations of the peptides, and both isomers of all peptide mimetics were assayed against S. aureus. Notably, peptides 2a and 2b were found to exhibit a four-fold difference between the cis-enriched and trans-enriched photostationary states. This study revealed a clear relationship between well-defined secondary structure, amphiphilicity, and optimal antibacterial activity. Chapter three describes the design and synthesis of three short photoswitchable tetrapeptides based on a known synthetic antibacterial. Each peptide contains an azobenzene photoswitch incorporated into either the N-terminal side chain, C-terminal side chain, or the C-terminus, to allow reversible switching between the cis and trans configurations. The C-terminus azobenzene (3) exhibited the most potent antibacterial activity against S. aureus, with an MIC of 1 μg/mL. A net positive charge, hydrophobicity, position of the azobenzene, secondary structure, and amphiphilicity were all found to be crucial to antibacterial activity. Chapter four reports a prodrug based on a known antibacterial compound to target S. aureus and E. coli under reductive conditions. The prodrug was synthesized by masking the N-terminus and side chain amines of a component lysine residue as 4-nitrobenzyl carbamates, while activation of the prodrug was achieved by removing the protecting groups under conditions that mimic hypoxia. Antibacterial susceptibility assays confirmed the liberation of the active antibacterial agent from the inactive prodrug to kill S. aureus and E. coli. Chapter five investigates the structural dynamic properties of a single-peptide using the single-molecule junction technique characterized by electrical conductance. The nanodevice was fabricated using a photoswitchable peptide containing an azobenzene photoswitch to provide both a well-defined secondary structure, and an intrinsically disordered structure. Real-time conductance measurements revealed three distinct states for each isomer, with molecular dynamics simulations showing each state corresponds to a specific range of hydrogen bond lengths within the cis isomer, and specific dihedral angles in the trans isomer. This study provides previously undisclosed insights into the structural dynamic behavior of peptides, which may well be applicable to proteins.